期刊
JOURNAL OF NEUROSCIENCE RESEARCH
卷 82, 期 6, 页码 858-865出版社
WILEY
DOI: 10.1002/jnr.20683
关键词
glutamate; tissue transglutaminase; astroglial cells; NF-kappa B inhibitor
We have previously demonstrated that alterations of cell redox state, evoked by glutamate, are associated with tissue transglutaminase increases in primary astrocyte cultures. Furthermore, glutamate exposure activated the nuclear factor (NF)-kappa B pathway, and its effects were significantly reduced by antioxidants. Here, we investigated the possible involvement of activated NF-kappa B pathway in glutamate-evoked tissue transglutaminase up-regulation in primary astrocytes. The presence of DNA binding activity by NF-kappa B in nuclear extracts of astrocytes, treated for 24 hr with glutamate (500 mu M) or untreated, was assessed by EMSA, using an oligonucleotide probe containing the NF-kappa B consensus sequence present in the tissue transglutaminase promoter. Supershifting with monoclonal antibodies revealed that activated NF-kappa B dimer complexes were composed of p50 and p65 subunits. Interestingly, the specific NF-kappa B inhibitor SN50 (but not its inactive analogue SN50M), when added to cell cultures 30 min prior to glutamate treatment, was able gradually to reduce glutamate-induced NF-kappa B activation. Western blot analysis confirmed the reduction of the p50 amount in nuclear extracts. Notably, the preincubation with SN50 also diminished glutamate-increased tissue transglutaminase expression, as showed by both RT-PCR and Western blotting. Competition experiments, carried out with an excess of a probe containing the NF-kappa B consensus sequence present in the kappa-light-chain promoter, demonstrated a preferential binding of the tissue transglutaminase specific NF-kappa B probe in the nuclear extracts of glutamate-treated astrocytes compared with untreated astrocytes. These preliminary data suggest that NF-kappa B activation, which has been demonstrated to be involved in astrocyte response to glutamate, could also be associated with the molecular pathway leading to glutamate-evoked tissue transglutaminase upregulation. (c) 2005 Wiley-Liss, Inc.
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